Self-flowing microfluidic analytical chip

ABSTRACT

A self-flowing microfluidic analytical chip may undergo spontaneous flow of a fluidic sample through microfluidic channels without an internal or external pump or corresponding pumping support hardware for fluid pumping. A self-flowing microfluidic analytical device includes sample preparation locations, sample analysis locations, and sample extraction locations connected by a network of microfluidic channels. Self-flowing characteristics of a microfluidic analytical chip result from maskless patterning of a substrate surface, where sequential passes of a patterning head preserve, rather than destroy, a pattern of surface functionalization. Self-flowing properties may be preserved by avoiding use of mask-removing solvents common to mask-removal steps in traditional microfluidic chip manufacturing processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent filing is a continuation of U.S. patent applicationSer. No. 15/600,492, titled SELF-FLOWING MICROFLUIDIC ANALYTICAL CHIP,filed 19 May 2017, which claims the benefit of U.S. Provisional PatentApplication 62/338,955, titled APPARATUS AND METHOD FOR PROGRAMMABLESPATIALLY SELECTIVE NANOSCALE SURFACE FUNCTIONALIZATION, filed 19 May2016; U.S. Provisional Patent Application 62/338,996, titled PUMP-FREEMICROFLUIDIC ANALYTICAL CHIP, filed 19 May 2016; U.S. Provisional PatentApplication 62/339,002, titled PUMP-FREE MICROFLUIDIC ANALYTICALSYSTEMS, filed 19 May 2016; and U.S. Provisional Patent Application62/339,008, titled STAND ALONE PUMP-FREE MICROFLUIDIC ANALYTICAL CHIPDEVICE, filed 19 May 2016. The content of each of these earlier filedpatent applications is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure related generally to a microfluidic analyticalchip (MAC) for testing fluids. Fluids may include biological fluidsadded to the microfluidic analytical chip for preparation, analysis, andprocessing. Biological fluid extraction and analysis may be utilized formedical diagnostics, identification, and testing. Conventional fluidextraction and analysis chips may be large and expensive. Self flowingmicrofluidic analytical chips may be less expensive to operate, morereliable, and provide greater availability of fluid analyte testingunder some analysis conditions. Self flowing microfluidic analyticalchips may be less expensive because self-flowing of a fluidic sampleacross the microfluidic chip means that external pumps, liquid supplies,gas supplies, and power supplies (for pumping) may be left out of amicrofluidic chip use method, reducing cost and simplifying testingprocedures. Microfluidic analytical chips may be used as part of an invitro diagnostics process or a point of care diagnostics method toidentify and resolve medical conditions, or to perform environmentaltesting for pathogens or other compounds.

BACKGROUND OF THE INVENTION

Microfluidic analytical chips may frequently be large and expensive toproduce. Large size and greater expense may be associated with the use,in traditional MACs, of large sample sizes in order to have sufficientanalyte for detection after sample preparation and processing. Reducingsample sizes while maintaining analytes within detectable concentrationranges of sensors compatible with a microfluidic analytical chip mayreduce LOC complexity and reduce costs associated with manufacturing andemploying MACs may be used in settings involving testing of fluidicsamples, including medical samples and environmental samples.

SUMMARY OF THE INVENTION

The invention addressing these and other drawbacks relates to methods,apparatuses, and/or systems for prioritizing retrieval and/or processingof data over retrieval and/or processing of other data.

Disclosed herein are embodiments of a chip that provides low cost,portable bio-fluid diagnostics. The chip includes a first substratehaving formed thereon microfluidic channels surface functionalized topromote self-flow of a fluid without any internal or external pumping.The chip further includes second substrate coupled to the firstsubstrate, providing cover for the microfluidic channels. Themicrofluidic channels may couple a sample extraction location, a samplepreparation location, and a sample analysis location. The sampleextraction location enables the fluid to be inserted into themicrofluidic channels.

The sample preparation location may include one or more preparationchambers, such as a reagent chamber for chemical reagent, membranechambers, a filters chamber, a micro heaters chamber, a fluid mixingchamber, a fluid separation chamber, and a waste collection chamber.

The sample analysis location may include one or more analysis chambers,such as an electrochemical analyte detection chamber utilizingelectrochemical analysis techniques, an optical analyte detectionchamber utilizing optical/florescence techniques, an enzyme analytedetection chamber utilizing enzyme-based detection, a columnchromatography analyte detection chamber utilizing column chromatographyin the microfluidic channels, and a spectrophotometry analyte detectionchamber utilizing fluorescent tagging.

Disclosed herein are embodiments of a microfluidic analytical chiphaving substrate with a pattern of surface functionalization with atleast two different types of surface functionalization therein, thesurface functionalization being configured to manipulate self flow of afluid across the surface of the substrate, and the pattern of surfacefunctionalization being formed by at least one maskless surfacefunctionalization process.

Also disclosed herein are embodiments of microfluidic analytical chipconfigured to analyze biological fluids or environmental samples.

These and other features of the present invention, as well as themethods of operation and functions of the related elements of structureand the combination of parts and economies of manufacture, will becomemore apparent upon consideration of the following description and theappended claims with reference to the accompanying drawings, all ofwhich form a part of this specification, wherein like reference numeralsdesignate corresponding parts in the various figures. It is to beexpressly understood, however, that the drawings are for the purpose ofillustration and description only and are not intended as a definitionof the limits of the invention. As used in the specification and in theclaims, the singular form of “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. In addition, asused in the specification and the claims, the term “or” means “and/or”unless the context clearly dictates otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated by way of example, and not by way oflimitation, in the figures of the accompanying drawing and in which likereference numerals refer to similar elements.

FIG. 1 illustrates an embodiment of a first substrate 100.

FIG. 2 illustrates an embodiment of microfluidic analytical chip havinga second substrate 200.

FIG. 3 illustrates an embodiment of sample preparation location 300.

FIG. 4 illustrates an embodiment of a testing device 400 configured toreceive a microfluidic analytical chip.

FIG. 5 illustrates an implementation of a method of analyzing a fluidsample using device microfluidic analytical chip 500.

FIG. 6 illustrates an implementation of a method of making an embodimentof a microfluidic analytical chip 600.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are embodiments of microfluidic analytical chip thatcomprise fluidic sample extraction, processing, and analysis. Fluidicsamples may include bodily fluids such as blood, saliva, sputum, andurine or environmental samples. Sample extraction may be accomplishedusing prick-free, touch based methods. Sample extraction may involve useof capillary action to draw a portion of a fluid (a fluid sample, orsample) into a sample extraction chamber prior to sample preparation orsample analysis. Sample preparation may involve mixing reagents with thesample, passing the combination of reagents and sample through filtersand membranes, heating the samples, separating blood cells and plasma,and other steps. Sample analysis may involve detection of variousbiomarkers by methods including electrochemical analysis of bloodcomposition (e.g., plasma vs serum), biomaterial detection usingoptical/florescence techniques, column chromatography in micro channels,flow cytometry, spectrophotometry using fluorescent tagging, andpotentially other techniques as well. Biomarkers may include antibodies,antigens, or other compounds associated with cellular metabolism or animmune response in an organism. Biological compounds detectable by amicrofluidic analytical chip may include components of pathogens thatcause illness, or components of cells indicative of illness of anorganism.

Referring to FIG. 1, a first substrate 100 of a microfluidic analyticalchip comprises first substrate first face 102, microfluidic channels104, sample extraction location 106, and sample analysis location 108.The first substrate 100 may be operated in accordance with the processesdescribed in FIG. 5 and FIG. 6.

In some embodiments, microfluidic channels 104 connect the sampleextraction location 106 and the sample analysis location 108. Themicrofluidic channels 104 may be etched or embossed into the firstsubstrate first face 102 or into a flat surface treated with functionalgroups, i.e., chemical moieties or specific groups of atoms or bondswithin molecules that are responsible for the characteristic chemicalreactions of those molecules. In embodiments in which the microfluidicchannels 104 are recessed below a major surface of the first substratefirst face 102, the microfluidic channels 104 may be formed by etchingor embossing the first substrate first surface 102. Microfluidicchannels in first substrate first face may have a channel width rangingfrom about 1000 micrometers (μm) down to about 100,000 micrometers (μm).A channel length of microfluidic channels may range from about 10centimeter (cm) to about 1,000 nm. A depth of a microfluidic channel mayrange from about 10 millimeters (mm) to about 5 Angstroms (Å), accordingto embodiments. The channel height may arise from raised materialartifacts of the embossing process. In an embodiment, the microfluidicchannels may be activated by adding a lyophilized compound to asubstrate first surface. Lyophilization may include rendering a reagentinto a powdered form prior to application of the lyophilized material toa substrate surface. In an embodiment, lyophilized chemical reagent maybe applied directly to an analyte detection surface in an analytedetection chamber. In an embodiment, a microfluidic pathway betweenlocations may be functionalized by reacting a lyophilized material witha plasma-processed surface of a microfluidic analytical chip.

In an embodiment, microfluidic channels may bedifferently-functionalized regions of a surface, rather than regionsthat extend above or below a major surface of a substrate surface. Adifferently functionalized portion of a major surface of a substratesurface may have a different degree of hydrophilicity than an unmodifiedportion of the substrate surface. In some embodiments, the first portionof the pattern may have greater degree of hydrophilicity than anunmodified portion of the first substrate first surface. In someembodiments, the first portion of the pattern may have greater degree ofhydrophobicity than an unmodified portion of the first substrate firstsurface. The different functionalization of the patterned portion of thesurface may induce fluids to move across the surface without pumping orpressurization to induce fluid motion across the surface. Capillaryaction, or a reduction of the surface tension of the fluid upon movementof the fluid to an uncovered portion of the differently functionalizedportion of the first substrate first surface, may draw a fluid across afirst substrate first surface, through openings in a substrate to adifferent level of a substrate, up conductive paths that extend above afirst substrate first surface, and onto a second substrate firstsurface.

The flow of fluid through the microfluidic channels 104 may be regulatedby control geometries on a substrate surface. Control geometries mayinclude breaks, or discontinuities, in the microfluidic channels 104 toreduce the speed of fluid flow. Control geometries may includeperforated sections to control the speed of the fluid flow. Controlgeometries may also include achieve metering of fluid flow by allowingfluid to accumulate at an “island” or “reservoir” before fluid proceedsthrough a microfluidic channel. Fluid may flow uniformly, or with astepwise change in flow rate, according to an embodiment. In a stepwiseflow scenario, fluid may proceed non-uniformly using flow controlgeometries to slow or to accelerate fluid flow through a circuit.

The microfluidic channels 104 may comprise mixing geometries to mixvarious fluids. The mixing geometries may comprise a series of S-shapedcurves in microfluidic channels 104, circular chambers with their inletaxis and outlet axis offset to allow eddies to form, and perforatedchannels to allow fluid to accumulate at an “island” and, once aspecified amount of fluid is collected, it may jump or hop to the nextsegment of the microfluidic channels 104.

The microfluidic channels 104 may be surface functionalized to deterevaporation of liquid from the microfluidic channels 104. The surfacemay be functionalized by coupling substrate material with functionalgroups configured to interact with components of a fluid flow.Functionalization of a substrate material may include addition offunctional groups to microfluidic channels to reduce an energy state ofthe liquid compared to an energy state of the liquid on a surfacewithout the functional groups, or a liquid separated from the substratesurface. A reduced energy state of a fluid may be associated with agreater attraction of the surface to the fluid, or to components of thefluid. Increased attraction may increase the energy associated withevaporation of the fluid from the substrate surface, deterring a rate ofevaporation of fluid from the surface.

In some implementations of microfluidic channels, a channel, or segmentsof a microfluidic channel, may have a varied channel dimension. Thevaried channel dimension may include channel height (or, depth), channellength, or channel width. The varied channel size may separate thecomponents in the fluid or filtering materials of a mixture in the fluidby utilizing the momentum difference among the components in the fluid.

The sample extraction location 106 may comprise a port to allowinsertion of fluid into the microfluidic channels 104. The sampleextraction location 106 may comprise one or more needles. The port mayfor example be formed from various materials, such as plastics, polymersor oxides. The port may be for example cylindrical, disc, or squareshape.

The sample extraction location 106 may comprise an array of microneedles to capture a fluidic sample from skin, tissues, or liquidsamples. Micro needles may penetrate through a membrane to access afluid, such as blood, lymph, or some other biological fluid. Microneedles may penetrate a surface of a fluid to reduce surface tension ofthe fluid and to promote entry of the fluid into sample extractionlocation. The array of micro needles may be formed from variousmaterials, such as silicon, oxides, crystalline materials, or compositematerials.

The sample analysis location 108 may comprise a combination of one ormore analysis chambers, including but not limited to multiple chambersof the same type. The sample analysis location 108 may in some casescomprise an electrochemical analyte detection chamber. Theelectrochemical analyte detection chamber may detect analytes in fluidsusing electrochemical analysis techniques. The electrochemical analytedetection chamber may comprise a first set of at least two electrodes.The first set of at least two electrodes may be printed orfunctionalized onto the first substrate first face 102 or onto thesecond substrate 200. In an embodiment, an electrode, or a pair ofelectrodes, may be formed at a surface of an electrochemical analytedetection chamber in order to measure an a flow of electrical currentthrough the fluidic sample In an embodiment, an electrode mayinterconnect through a body of a substrate, to a conductive pad at aremove form an electrochemical analyte detection chamber in order topromote an electrical connection between the electrode and a signalrecording element configured to receive and analyze a signal from theelectrode.

The sample analysis location 108 may comprise an optical analytedetection chamber. The optical analyte detection chamber may detectanalytes in fluids using an optical/florescence technique. The opticalanalyte detection chamber may use light transmitted through the firstsubstrate or the second substrate as either the first substrate or thesecond substrate may be formed from materials that may transmit light.

The sample analysis location 108 may comprise a biomaterial analytedetection chamber. The biomaterial analyte detection chamber may detectanalytes in fluids using enzyme-based detection techniques or othertechniques using biomaterials to trigger chemical changes in a fluidicsample. In an embodiment, biomaterials may trigger a signal by changinga color of a solution, by consuming an analyte to produce a detectablereaction product, or to produce a compound configured to adhere to anelectrode surface and modify a signal from an analyte chamber. In anembodiment, biomaterials may be functionalized onto the surface of thefirst substrate or the second substrate to promote a chemical changethat may be subsequently detected. In an embodiment, detection of achemical change may include optical detection of a product of a chemicalreaction.

The sample analysis location 108 may comprise a column chromatographyanalyte detection chamber. The column chromatography analyte detectionchamber may detect analytes in fluids using column chromatography in themicrofluidic channels 104. Chromatographic material may befunctionalized onto the first substrate first face, or onto the secondsubstrate in order to retain components of the fluidic sample duringpassage through fluidic channel. An analyte detection chamber may beconfigured to respond to a functional group on an analyte by binding theanalyte and modifying a voltage of an electrode, or by binding ananalyte and modifying a fluorescent taggant functionalized to theanalyte detection chamber surface, or some other method of selectingamong analyte fractions following a chromatographic process.

The sample analysis location 108 may comprise a spectrophotometryanalyte detection chamber. The spectrophotometry analyte detectionchamber may detect analytes in fluids using spectrophotometry, forexample, by fluorescent tagging. The spectrophotometry analyte detectionchamber may utilize light transmitted through the first substrate or thesecond substrate.

FIG. 2 depicts a microfluidic analytical chip 200 having a secondsubstrate 201 comprising a second substrate first face 202. Themicrofluidic analytical chip 200 may be operated in accordance with theprocesses described in FIG. 5 and FIG. 6.

The second substrate 200 may further interact with first substrate 100,first substrate first face 102, and microfluidic channels 104. In someembodiments, the second substrate 201 with second substrate first face202 may be placed on first substrate first face 102 of first substrate100 to provide a cover for microfluidic channels 104. The secondsubstrate first face 202 may be located between the first substratefirst face 102 and the body of the second substrate. According to anembodiment, the second substrate comprises a material having a lowdegree of autofluorescence. In an embodiment with low autofluorescencesecond substrate material, optical signals form analytes passing throughoptical detection chambers may be detected at lower concentrations ofanalyte than in embodiments with second substrates having larger degreesof autofluorescence. A determination between a low-autofluorescence or ahigh-autofluorescence second substrate may relate to a cost ofmanufacturing device microfluidic analytical chip, an array of analytetesting chambers configured on a first substrate, and an anticipatedamount of signal for an analyte according to a predicted usage scenarioof a microfluidic analytical chip.

The first substrate 100 may have a first substrate material wherein thedegree of hydrophilicity (or, hydrophobicity) of the surface may bemodified by plasma-based surface functionalization. The first substratemay have a first substrate first surface made of a plastic material, apolymer, an inorganic oxide, such as silicon dioxide,polydimethylsiloxane (PDMS), or glass, inter alia. The second substrate201 may have a plastic material, a polymer, an inorganic oxide, such assilicon dioxide, polydimethylsiloxane (PDMS), or glass, inter alia. Thefirst substrate 100 and the second substrate 200 may be formed from thesame material. Alternatively, the first substrate 100 may be formed froma different material than the second substrate 201.

Referring to FIG. 3, the sample preparation location 300 may comprisereagent chamber 302, membrane chambers 304, filters chamber 306, microheaters chamber 308, electrodes chamber 310, fluid mixing chamber 312,fluid separation chamber 314, and waste collection chamber 316. Thesample preparation location 300 may be operated in accordance with theprocesses described in FIG. 5 and FIG. 6.

In some embodiments, sample preparation location 300 is located on thefirst substrate 100. The sample preparation location 300 may be coupledto the sample extraction location 106 and the sample analysis location108 via the microfluidic channels 104. The sample preparation location300 may comprise a combination of one or more preparation chambers. Thesample preparation location 300 may comprise multiple chambers of thesame type. Sample preparation location may comprise multiple chamberswith different types. Chambers of the sample preparation location may belocated, with respect to each other, in series, in parallel, or incombinations of series and parallel arrangements, in order to providereagents to a fluid stream during sample preparation and prior to sampleanalysis.

The reagent chamber 302 may store a reagent, for example, a chemicalreagent. The reagent may be stored or delivered through the microfluidicanalytical chip using passive valves. Passive valves may allow or delayflow of a fluid by virtue of the geometry of the passive valve and thepattern of surface functionalization through a portion of the channelsof the microfluidic analytical chip. by way of at least one flowregulating valve, or shot valve. The at least one passive flowregulating valve may be located on the first substrate first face or onthe second substrate. The at least one passive flow regulating valve maydisrupt the microfluidic channels 104. Fluid may also be retained in achamber by special functionalization of the walls of the chamber,wherein the special functionalization is configured to reduce a surfacetension of the fluid within the chamber and to retain a portion of fluidwithin a chamber against a portion of a functionalized surface withinthe chamber. Special functionalization may include polar functionalgroups connected to a chamber wall, wherein the polar groups of thespecial functionalization may attract and retain water put in proximityto the walls.

Substrate services may be functionalized using a pattern to protect afirst portion of a substrate surface while a second portion of substratesurface is exposed to functional icing conditions. In traditionalmasking processes, layer of photoresist may be deposited on a substratetop surface, and layer of photoresist may be patterned, such as byultraviolet light, and developed in order to generate regions of themasking layer where the substrate surface is exposed. In a non-limitingembodiment, a polymethylmethacrylate (PMMA) substrate may be coated witha layer of photoresist. The photoresist may be patterned by exposing thephotoresist to ultraviolet light, followed by a developing step to rinsea portion of the photoresist layer off of the substrate surface. Theexposed portion of the substrate service may be functionalized byexposing the exposed portion to a plasma may modify his chemistrysubstrate in the exposed area, resulting in a modified chemical orphysical characteristic of the substrate in the exposed portion.However, in order to remove the patterned photoresist from the substratetop surface, chemical treatments such as isopropyl alcohol (IPA),acetone, or alcohols may, while removing the photoresist, also removesome or all of the functionalization formed on the substrate surface.Thus, the functionalized chemistry may be incompatible with solvents orthe chemical makeup of a functionalized surface after a plasmaprocessing step to functionalize a substrate.

Functionalizing chemistry may include radical species or non-radicalspecies that result in the addition of oxygen to a substrate surface. Arepresentative sample of compounds that may generate an oxygen-modifiedsurface may include a substrate

According to an embodiment, a supply of fluid (a gas or a liquid) to theworking volume during surface modification may adjust the chemicalcomposition of the substrate top surface during the surface modificationprocess. According to an embodiment, a fluid mixture may include one ormore gaseous species, or may include a volatilized (or aerosolized)liquid that, upon evaporation, provides a gaseous component for the gasmixture.

Chemical species that may be used for surface functionalization includecompounds for increasing a concentration of surface oxygen on asubstrate surface, compounds for increasing a concentration of a halogenon a substrate surface, and compounds for increasing a concentration ofnitrogen on a substrate surface. Chemical species that functionalize asurface may be radicals or nonradicals. Chemical species that maypromote functionalization of a surface with halogen atoms, includingchlorine or bromine, may include atomic chlorine or atomic bromine, ornon-radical species such as: hypochlorous acid (HOCl), nitryl chloride(NO₂Cl), chloramines, chlorine gas (Cl₂), bromine chloride (BrCl),chlorine dioxide (ClO₂), hypobromous acid (HOBr), or bromine gas (Br₂).Chemical species related to addition of oxygen to a substrate surfacemay include radicals or non-radical species, such as: superoxide (O₂ ⁻),hydroxyl radicals (HO.), hydroperoxyl radical (HO₂.), carbonate (CO₃.⁻),peroxyl radicals (RO₂.), where R is a carbon or other atom, and alkolxylradicals (RO.), where R is a carbon or other atom, as well as nonradicalspecies such as hydrogen peroxide, hypobromous acid (HOBr), hypochlorousacid (HOCl), ozone (O₃), organic peroxides (ROOH), where R=C,poroxynitrite (ONOO⁻), or peroxynitrous acid (ONOOH). Chemical speciesrelated to addition of nitrogen to a substrate surface may includespecies such as nitric oxide NO., nitrogen dioxide NO₂., nitrate radical(NO₃.), nitrous acid (HNO₂), dinitrogen tetroxide (N₂O₄), dinitrogentrioxide (N₂O₃), peroxynitrite (ONOO⁻), peroxynitrous acid (ONOOH), ornitryl chloride (NO₂Cl).

The membrane chambers 304 may be coupled to the first substrate firstface 102, for example, by functionalizing the surface withmembrane-binding chemistries to couple membrane materials to the firstsubstrate 100. The membrane chambers may also be coupled to the secondsubstrate 200, for example, by functionalizing the surface withmembrane-binding chemistries to couple membrane materials to the secondsubstrate 200. The filters chamber 306 may comprise filters. The filtersmay be coupled to the first substrate first face 102 by functionalizingthe surface with filter-binding chemistries that allows binding of thefilters to the first substrate 100. The filters chamber 306 may also becoupled to the second substrate 200, for example, by functionalizing thesurface with filter-binding chemistries that allows binding of thefilters to the second substrate 200.

Binding chemistries for filters or membranes of a microfluidic deviceanalytical chip may include chemical moieties that attract a portion ofa membrane or a filter to an activated portion of a substrate face. Inan embodiment, a filter or membrane may be retained in a chamber of themicrofluidic analytical chip by a fluid flow, or by an adhesive. In someembodiments, chemical moieties may be reversibly binding. In someembodiments, chemical moieties may be permanently binding, such that afilter or membrane is permanently attached to the substrate face.

According to an embodiment, a reversible binding chemistry may includeformation of complexes or intermolecular clusters. In an embodiment,binding chemistry may include one or more sets of deoxyribonucleic acid(DNA) base pairs configured, by inclusion of one nucleobase of a DNAbase pair, to attract and retain a portion of a filter or a membrane toan activated portion of a substrate face (e.g., having the other,complimentary nucleobase, of the DNA base pair). Pairs of nucleobasesmay form hydrogen bonds that retain one nucleobase against anothernucleobase. In an embodiment, a rectangular membrane material may beconfigured with the nucleobase adenine on a first edge of the membranematerial, and with guanine on a second edge of the membrane material,the second edge being opposite the first edge. A first substrate mayhave an activated region with the nucleobase thymine at a position inthe microfluidic circuit where the membrane should attach to perform afiltering function, and a second substrate may have the nucleobasecytosine at a position in the microfluidic circuit where the membraneshould attach. In an embodiment, the functionalization of substratefaces may be performed such that the membrane is bound to a singlesubstrate face, with complimenting base pair interactions occurring atthe sides of a microfluidic channel wherein the membrane is positioned.In an embodiment, a pattern of nucleobase activation (functionalization)on a substrate face may be configured to orient a membrane with a firstorientation to present a first membrane face in an upstream orientation(i.e., toward a source for a fluid that traverses the microfluidicchannel during operation of the microfluidic device).

In an embodiment, a permanent binding chemistry may include adehydration reaction, or a condensation reaction between two reactantsfunctionalized onto a substrate face and a membrane or filter materialfor a microfluidic circuit. In an embodiment, a condensation reactionconfigured to bind a membrane or a filter material within a channel of amicrofluidic device or an integrated testing device may include [1]condensation of amino acids to form peptide bonds, or [2] a condensationreaction between a carboxylic acid and an alcohol to form an ester.Other examples of functionalization appropriate for binding chemistriesof membranes or filters may be known to a practitioner of reasonableskill in the arts. A low-power plasma processing method that canactivate, in a non-destructive manner, a surface of a material forfunctionalization, may allow configuration of membrane or filtermaterials with customizable reversible or permanent binding chemistriesto retain membranes or filters, including both synthetic andnaturally-occurring membrane or filter materials, in microfluidicchannels.

In an embodiment, a filter may be configured to trap particles orcomponents of a fluid that exceed a threshold size of a filter opening.In an embodiment, a plurality of filters may be used, in series, or inparallel, or in pluralities of filtering chambers, to fractionate afluid during an analysis process.

The micro heaters chamber 308 may comprise micro heaters. The microheaters may be located on the first substrate 100, or may be located onthe second substrate 200. A micro heater chamber may be located in asubstrate body to regulate a temperature of a microfluidic channelduring analysis of a fluid.

The electrodes chamber 310 may comprise electrodes that may perform anelectrochemical process. The electrodes may be printed on one or more ofthe first substrate 100 and the second substrate 200 by anelectrode-printing technique. A combination of electrodes on one or moreof the first and second substrates may allow for improved detection ofanalytes in an analytic chip or integrated testing device.

The fluid mixing chamber 312 may mix fluids, and may have a chambergeometry to help develop eddy currents to mix fluid mixing chamberfluids. Mixing of fluids in a microfluidic device may include, accordingto some embodiments, chambers with asymmetric entry and exit locationswith regard to a center of the mixing chamber. An asymmetric fluid paththrough a mixing chamber may induce an eddy current, or rotationalmotion of the fluid around a center of the mixing chamber, wherein sucheddy currents or rotational motion cause blending between one or morecomponents of the fluid stream into the mixing chamber. In anembodiment, mixing of a fluid stream may serve to promote uniformdistribution of solutes through the fluid stream. In an embodiment,mixing of a fluid stream may serve to promote a uniform distribution ofa suspended material in the fluid stream.

The fluid separation chamber 314 may comprise separation channelsconfigured to separate components a fluidic mixture. The separationchannels may have an alterable channel width to regulate a flow velocitythrough the separation channel. Varying a flow velocity of the fluidthrough the separation channel may separate the fluidic mixture intofluidic mixture components. In an embodiment, a flow rate of a fluidthrough a narrow portion of the separation channel may be greater than afluid flow rate through a wider portion of the separation channel. Areduction in fluid flow rate may be associated with fluid componentshaving a larger mass or molecular weight moving along the separationchannel more slowly than lower mass, or smaller molecular weight, fluidcomponents.

The waste collection chamber 316 may allow the flow of fluids in thenetwork of microfluidic channels 104. In some embodiments, one or morewaste collection chambers are sized to receive a flow of fluid throughmicrofluidic channels through a testing and analysis process, wherein,when one waste collection chamber fills with fluid, a second wastecollection chamber may continue to fill with fluid to promote continuousand even fluid flow throughout a fluid preparation and analysis process.

FIG. 4 depicts a testing device 400 configured to receive a microfluidicanalytical chip. The analytical chip may comprise a sample extractionlocation 106, a sample analysis location 108, a sample preparationlocation 300, and a sample data process and transmission 402. The fluidanalytical device 400 may be manufactured and operated in accordancewith the processes described in FIG. 5 and FIG. 6 and be configured toanalyze, inter alia, components of a biological fluid or suspension. Inan embodiment, fluidic analytical device 400 may include One embodimentof an analytical device may include a plurality of microfluidic channelsconfigured to separate and analyze blood serum or blood plasma. Oneembodiment of an analytical device may include a plurality ofmicrofluidic channels configured to separate and analyze components ofenvironmental samples for contamination or biological activity. Oneembodiment of an analytical device may include a plurality ofmicrofluidic channels configured to separate and analyze pathogens orbiomarkers of disease.

In some embodiments, an fluid analytical device 400 may comprise acombination of at least two locations, the at least two locations chosenfrom the sample extraction location, the sample preparation location,and the sample analysis location. The sample data process andtransmission 402 may be electronically coupled to the sample analysislocation 108 and receive signals from the sample analysis location 108.The received signal may comprise analysis information obtained from thefluid analyzed in the sample analysis location 108. The sample dataprocess and transmission 402 may transmit the signals to one or moremachines. The transmission may occur via a wired or wireless connectionto the one or more machines, for example via a machine data network.

FIG. 5 depicts an implementation of a method of analyzing a fluid sampleusing device microfluidic analytical chip 500 to identify a component ofthe fluidic sample. Method 500 includes an operation 502 wherein a fluid(fluidic sample) is extracted from a fluid source to the sampleextraction location prior to manipulation of the fluid through theintegrated testing device.

Method 500 includes an operation 504, wherein a flow of the fluid isdirected from the sample extraction location to a sample preparationlocation. Method 500 includes an operation 506, the fluidic sample isprepared in the sample preparation location. Sample preparationlocations in an integrated testing device may include a reagent chamberfor a chemical reagent, a membrane chamber, a filters chamber, a microheater chamber, a fluid mixing chamber, a fluid separation chamber, anda waste collection chamber. Sample preparation may include, for onevolume of a fluidic sample prepared by the integrated testing device,preparation in at least one of the sample preparation chambers listedhereinabove. In some embodiments, a volume of a fluidic sample may beprocessed through multiple sample preparation chambers in order toprepare the sample for analysis.

Method 500 includes an operation 508, the flow of the fluidic sample isdirected from the sample preparation location to a sample analysislocation. Method 500 includes an operation 510, the fluidic sample isanalyzed at the sample analysis location. Analysis of a sample mayinclude direction of a volume of fluidic sample toward, and observationof the fluidic sample within, at least one of an electrochemical analytedetection chamber, the electrochemical analyte detection chamber usingelectrochemical analysis techniques; an optical analyte detectionchamber, the optical analyte detection chamber using optical/florescencetechniques; an enzyme analyte detection chamber, the enzyme analytedetection chamber using enzyme-based detection; a column chromatographyanalyte detection chamber, the column chromatography analyte detectionchamber using column chromatography in the microfluidic channels; and anspectrophotometry analyte detection chamber, the spectrophotometryanalyte detection chamber using fluorescent tagging.

Method 500 includes an operation 512, a signal is transmitted from thesample analysis location to a sample data process and transmissionstage. Method 500 includes an operation 514, the signal from the sampledata process and transmission stage is transmitted to one or moremachines. In some embodiments, the flow of the fluidic sample isdirected by microfluidic channels 104.

In a representative, non-limiting embodiment of the method of analyzinga fluid using an integrated testing device, a fluidic sample may beextracted from a fluid source and introduced into a sample extractionlocation, wherein the fluidic sample is a volume of blood plasma takenfrom a patient with an infection. According to the non-limitingembodiment of the method 500, the volume of blood plasma may be directedto a sample preparation location wherein the fluidic sample may beprepared by perform a series of preparation operations thereon,including, for example, filtering of a volume of the fluidic sample toisolate a pathogen. Sample filtration may be performed by passing thevolume of the fluid sample through a membrane having openings with athreshold size, allowing some components of the fluidic sample to passthrough, while retaining other components of the fluidic sample behindthe membrane. A membrane of the integrated testing device wherein thenon-limiting embodiment of the method may be may be configured such thatblood cells of the fluidic sample are retained at the membrane, whilepathogens non-cellular components of the fluidic sample pass through themembrane. Sample preparation may further include a processing stepwherein a chemical reagent, including a first type of fluorescent dyemolecule, with a first type of chemical binding component, may beintroduced to the volume of fluidic sample, mixing the fluorescent dyemolecule with the pathogen. Of a plurality of pathogen-binding chemicalbinding components of the sample preparation location, the first type offluorescent dye molecule may label pathogens having a binding sitecorresponding with the first type of chemical binding component.

The labeled pathogens of the volume of fluidic sample may be furtherdirected toward a sample analysis location. Sample analysis locationsmay include least one of an electrochemical analyte detection chamber,the electrochemical analyte detection chamber using electrochemicalanalysis techniques; an optical analyte detection chamber, the opticalanalyte detection chamber using optical/florescence techniques; anenzyme analyte detection chamber, the enzyme analyte detection chamberusing enzyme-based detection; a column chromatography analyte detectionchamber, the column chromatography analyte detection chamber usingcolumn chromatography in the microfluidic channels; and anspectrophotometry analyte detection chamber, the spectrophotometryanalyte detection chamber using fluorescent tagging. Volumes of fluidicsample having been processed with fluorescent dye molecules, may bedirected to, e.g., a spectrophotometry analyte detection chamber foroptical testing. The volumes of fluidic samples may be exposed to anillumination source configured to promote fluorescence of moleculesbound to pathogens or to components of pathogens. Light emitted duringfluorescence of bound pathogens and/or bound pathogenic components maybe detected by an optical detection bench and a signal transmitted to adata analysis component of the integrated testing device.

In a further representative, non-limiting embodiment of the presentdisclosure, a fluid sample may include a population of cells carrying agenetic marker or a genetic mutation indicative of an illness. A samplepreparation operation may include filtering the fluid to collect thepopulation of cells carrying the genetic marker or genetic mutation,cleaving cell walls to expose an interior of the cells having thegenetic marker or mutation, and blending the cleaved cells with asolution containing mixture of chemicals, such as nucleobases and PCR,configured to amplify a number of strands of DNA. Sample preparation mayalso include treatment of the fluidic solution, or the ruptured cellswithin the fluidic solution, with a mixture of compounds configured tounzip DNA or to cleave DNA into fragments for testing purposes.According to an embodiment, a sample analysis operation may include,subsequent to binding fluorescent taggant molecules to DNA strands orfragments containing the genetic marker or genetic mutation, performingan optical analytical process (e.g., fluorescence spectrophotometry) todetermine a number of bound DNA fragments having the genetic marker orgenetic mutation (or, e.g., a presence of the genetic marker or geneticmutation in the fluidic sample after the sample preparation operation.

FIG. 6 depicts an implementation of a method of making an embodiment ofa microfluidic analytical chip 600. Method 600 includes operation 602, asample extraction location is formed on a first substrate. In operation604, a sample preparation location is also formed on the firstsubstrate. In operation 606, the sample preparation location is coupledto the sample extraction location. In operation 608, a sample analysislocation is formed on the first substrate. In operation 610, the sampleanalysis location is coupled to the sample preparation location. Inoperation 612, a sample data process and transmission stage is coupledto the sample analysis location. In operation 614, a second substratemay be combined with the first substrate to form a cover formicrofluidic channels in an analytic chip or integrated testing device.Inclusion of a second substrate may reduce evaporation of a sampleduring analysis, provide optical windows for sample analysis, andmaintain sterility or cleanliness of the microfluidic channels prior tointroduction of a fluidic sample to the microfluidic channels. In doneoperation 616, the method 600 ends.

The microfluidic channels may be etched into the first substrate, orembedded into the first substrate. The microfluidic channels may beutilized to couple the sample extraction location, the samplepreparation location, and the sample analysis location.

In embodiments utilizing etching, the microfluidic channels may have achannel width ranging from 1000 microns to 100 nanometers, a channellength ranging from 10 centimeter to 100 nanometers, and a channel depthranging from 1 millimeter to 5 angstroms. In embodiments utilizingembossing, the microfluidic channels may a channel width ranging from1000 microns to 100 nanometers, a channel length ranging from 10centimeter to 100 nanometers, a channel depth ranging from 1 millimeterto 5 angstroms, and a channel height ranging from 1 millimeter to 5angstroms.

The microfluidic channels may be formed with control geometries (e.g.,breaks and dotted sections) to control a flow of the fluid. Themicrofluidic channels may be formed with mixing geometries to mixvarious fluids, for example serpentine structures, offset inlets in acircular location to allow swirling, and dotted channels. Themicrofluidic channels may be surface functionalized to deter evaporationof liquid from the microfluidic channels, so that liquid in themicrofluidic channels has a lower energy state than in an evaporatedstate in air. The microfluidic channels may be formed with a variedchannel size to separate components in the fluid or filtering materialsof a mixture in the fluid.

The sample extraction location may be formed with a needle insertionport to allow insertion of liquids, the needle insertion port composedof plastics, polymers, or oxides and cylindrical, disc or square shape.The sample extraction location may be formed with an array of microneedles to capture fluidic sample from skin or tissues or liquidcontaining features, for example from oxides, crystalline materials likesilicon or oxides or composite materials like polymer and nanomaterials.

The reagent chamber may be formed to include least one shot valve or aspecific functionalization of reagents on the first substrate first faceor valves created by disrupting the microfluidic channels or the secondsubstrate. The membrane chambers may be coupled to the first substratefirst face by functionalizing the surface with membrane-bindingchemistries to couple membrane materials to the first substrate or thesecond substrate. The filters chamber may be formed to include filterscoupled to the first substrate first face by functionalizing the surfacewith filter-binding chemistries that allows binding of the filters tothe first substrate or the second substrate. The micro heaters chambermay be formed to include micro heaters located in the first substrate orthe second substrate. The electrodes chamber may be formed to includeelectrodes printed on the first substrate or the second substrate by anelectrode-printing functionalization technique. The fluid mixing chambermay be formed to include fluid mixing geometries to develop eddycurrents to mix fluid mixing chamber fluids, and the fluid separationchamber may be formed with separation channels having a fluidic mixture,the size of the separation channels alterable to separate the fluidicmixture into fluidic mixture components.

The electrochemical analyte detection chamber may be formed with a firstset of at least two electrodes printed or functionalized on the firstsubstrate first face or the second substrate. The optical analytedetection chamber may be formed to utilize light transmitted through thefirst substrate or the second substrate, and the enzyme analytedetection chamber may be formed with enzymes functionalized on thesurface of the first substrate or the second substrate. The columnchromatography analyte detection chamber may be formed from achromatographic material functionalized onto the first substrate firstface or the second substrate. The spectrophotometry analyte detectionchamber may be formed to utilize light transmitted through the firstsubstrate or the second substrate.

A combination of at least two locations chosen from the sampleextraction location, the sample preparation location, and the sampleanalysis location may form the fluid analytical device.

In some embodiments, the analytical chip described herein may bemanufactured with a patterning device like that described in a U.S.patent application titled APPARATUS AND METHOD FOR PROGRAMMABLESPATIALLY SELECTIVE NANOSCALE SURFACE FUNCTIONALIZATION filed on thesame day as this patent filing, the contents of which are incorporatedby reference. In some embodiments, the analytical chip may be analyzedwith a self-flowing microfluidic analytical system described in a U.S.patent application tiled STAND ALONE MICROFLUIDIC ANALYTICAL CHIPDEVICE, filed on the same day as the present patent filing, the contentsof which are incorporated by reference.

What is claimed is:
 1. A device comprising a first substrate having: afirst substrate first face; a plurality of microfluidic channels on thefirst substrate first face and being surface functionalized forself-flowing fluid manipulation, and being connected to: a sampleextraction location, a sample preparation location, and a sampleanalysis location; wherein the sample extraction location beingconfigured to direct a fluid, received at the sample extractionlocation, into the plurality of microfluidic channels; the samplepreparation location having one or more preparation chambers comprisingat least one of a reagent chamber for a chemical reagent, a membranechamber, a filters chamber, a micro heater chamber, a fluid mixingchamber, a fluid separation chamber, and an optical fluorescencechamber, and a waste collection chamber; and the sample analysislocation having one or more analysis chambers including at least one of:an electrochemical analyte detection chamber, the electrochemicalanalyte detection chamber using electrochemical analysis techniques; anoptical analyte detection chamber, the optical analyte detection chamberusing optical/florescence techniques; a biomaterial analyte detectionchamber, the biomaterial analyte detection chamber usingbiomaterial-based detection; a column chromatography analyte detectionchamber, the column chromatography analyte detection chamber usingcolumn chromatography in the microfluidic channels; and aspectrophotometry analyte detection chamber, the spectrophotometryanalyte detection chamber using fluorescent tagging.
 2. The device ofclaim 1, further comprising a second substrate having a second substratefirst face and a second substrate body, the second substrate providing acover for the microfluidic channels, the second substrate first facebeing between the first substrate first face and the second substratebody.
 3. The device of claim 2, wherein: the first substrate comprises afirst substrate material, the first substrate material made of a firstpolymer, a first plastic, or a first inorganic oxide; and wherein thesecond substrate comprises a second substrate material, the secondsubstrate material made of a second polymer, a second plastic, or asecond inorganic oxide.
 4. The device of claim 1, wherein: the pluralityof microfluidic channels are recessed channels below a major surface ofthe first substrate first face, wherein individual microfluidic channelsof the plurality of microfluidic channels have: a channel width of atleast 100 nanometers and not greater than 100,000 micrometers; a channellength of at least 100 nanometers and not greater than 1,000centimeters; and a channel depth of at least 5 angstroms and not greaterthan 10 millimeter.
 5. The device of claim 4, wherein the plurality ofmicrofluidic channels are recessed below a major surface of the firstface of the first substrate by etching or embossing the major surface offirst substrate first face.
 6. The device of claim 1, wherein theplurality of microfluidic channels comprise control geometries tocontrol a flow of the fluid, the control geometries comprising breaksand dotted sections.
 7. The device of claim 1, wherein the plurality ofmicrofluidic channels comprise mixing geometries to mix various fluids,the mixing geometries comprising at least serpentine structures, offsetinlets in a circular location to allow swirling, and dotted channels. 8.The device of claim 1, wherein the plurality of microfluidic channelsare further surface functionalized to deter evaporation of liquid fromthe microfluidic channels, the liquid in the microfluidic channelshaving a lower energy state than in an evaporated state in air.
 9. Thedevice of claim 1, wherein at least one microfluidic channel has avaried channel size configured to separate at least two component of thefluid.
 10. The device of claim 1, wherein the sample extraction locationfurther comprises a needle insertion port comprising a polymer, aplastic, or an oxide.
 11. The device of claim 1, wherein the sampleextraction location further comprises an array of micro needles toreceive fluid from skin or a fluid-filled feature, the array of microneedles comprising composite materials such as polymers and nanomaterials, or crystalline materials such as silicon and an inorganicoxide.
 12. The device of claim 1, wherein: the chemical reagent isretained in the reagent chamber by at least passive valve or a specificfunctionalization of reagents on the first substrate first face orvalves created by disrupting the microfluidic channels or the secondsubstrate first surface; the membrane chambers are coupled to the firstsubstrate first face by functionalizing the surface withmembrane-binding chemistries to couple membrane materials to at leastone of the first substrate or the second substrate; the filters chambercomprise filters, the filters coupled to the first substrate first faceby functionalizing the surface with filter-binding chemistries thatallows binding of the filters to at least one of the first substrate orthe second substrate; the micro heaters chamber comprise micro heaters,the micro heaters located in the first substrate or the secondsubstrate; the electrodes chamber comprising electrodes, the electrodesprinted on the first substrate or the second substrate by anelectrode-printing functionalization technique; the fluid mixing chambercomprising fluid mixing geometries to develop eddy currents to mix fluidmixing chamber fluids; and the fluid separation chamber comprisesseparation channels, the separation channels containing a fluidicmixture, the size of the separation channels alterable to separate thefluidic mixture into fluidic mixture components.
 13. The device of claim1, wherein: the electrochemical analyte detection chamber comprises afirst set of at least two electrodes printed or functionalized on theface of one or more of the first substrate and the second substrate; theoptical analyte detection chamber uses light transmitted light throughthe first substrate or the second substrate; the enzyme analytedetection chamber comprises enzymes functionalized on the surface of thethe first substrate or the second substrate; the column chromatographyanalyte detection chamber comprises a chromatographic material, thechromatographic material functionalized onto the first substrate firstface or the second substrate; and the spectrophotometry analytedetection chamber uses light transmitted through the first substrate orthe second substrate.
 14. The device of claim 1, wherein a combinationof at least two locations comprises a fluid analytical device, the atleast two locations chosen from the sample extraction location, thesample preparation location, and the sample analysis location.
 15. Amicrofluidic device comprising: a substrate having a first substrateface, the first substrate face having an unmodified area and a patternedarea, the unmodified area having an unmodified surface functionalizationand the patterned area having at least one other type of surfacefunctionalization configured to promote self flow of a fluid, withoutpumping, across the first substrate first surface, wherein at least oneof the first portion of the patterned area, having a first type ofsurface functionalization, and a second portion of the patterned area,having a second type of surface functionalization, is formed by at leastone maskless surface functionalization process.
 16. The microfluidicdevice of claim 15, wherein at least one of the first type of surfacefunctionalization and the second type of surface functionalization is atype of functionalization that is removed from a surface when a maskmaterial is removed.
 17. The microfluidic analytical chip of claim 15,wherein at least some of the patterned area further compriseslyophilized chemical pathways.
 18. A arrangement comprising a firstsubstrate having: a first substrate first face; a plurality ofmicrofluidic channels on the first substrate first face and beingsurface functionalized for self-flowing fluid manipulation, and beingconnected to: a sample extraction location, a sample preparationlocation, and a sample analysis location; wherein the sample extractionlocation being configured to direct a fluid, received at the sampleextraction location, into the plurality of microfluidic channels; thesample preparation location having one or more preparation chamberscomprising at least one of a reagent chamber for a chemical reagent, amembrane chamber, a filters chamber, a micro heater chamber, a fluidmixing chamber, a fluid separation chamber, and an optical fluorescencechamber, and a waste collection chamber; and the sample analysislocation having one or more analysis chambers including at least one of:an electrochemical analyte detection chamber, the electrochemicalanalyte detection chamber using electrochemical analysis techniques; anoptical analyte detection chamber, the optical analyte detection chamberusing optical/florescence techniques; a biomaterial analyte detectionchamber, the biomaterial analyte detection chamber usingbiomaterial-based detection; a column chromatography analyte detectionchamber, the column chromatography analyte detection chamber usingcolumn chromatography in the microfluidic channels; and aspectrophotometry analyte detection chamber, the spectrophotometryanalyte detection chamber using fluorescent tagging.